Differential counting of leukocytes and other cells

ABSTRACT

A mixture of dyes for differentially staining biological cells. A system for the automatic absolute counting of leukocytes, erythrocytes and reticulocytes and differential counting of leukocytes utilizes a composition characterized by specific fluorescent dyes for staining a blood specimen and includes means for irradiating, counting and classifying leukocyte types. When the stained sample is irradiated, characteristic fluorescent intensities in specific spectral ranges uniquely identify particular leukocyte types.

United States Patent Kleinerman DIFFERENTIAL COUNTING OF LEUKOCYTES AND OTHER CELLS 3,497,690 2/1970 Wheeless, Jr. et al 250/304 [75] Inventor: Marcos Kleinerman, Arlington, Primary ExaminerArchie R. Borchelt Mass. Attorney, Agent, or FirmSchiller & Pandiscio [73] Assignee: Block Engineering, Inc., Cambridge, Mass 57 ABSTRACT [22] Filed: May 31, 1973 I A mixture of dyes for differentially staining biological [21] Appl' 365460 cells. A system for the automatic absolute counting of leukocytes, erythrocytes and reticulocytes and differ- [52] US. Cl. 250/461; 250/302; 250/304; ential n ing f l yte lize a comp ition 356/36; 356/39 characterized by specific fluorescent dyes for staining [51] Im. Cl. G01N 21/38 a blood Specimen and includes means for irradiating, [58] Field of Search 250/302, 304, 365, 458, o n ng an classifying leukocyte types. When the 250/461; 3 56/36, 39 stained sample is irradiated, characteristic fluorescent intensities in specific spectral ranges uniquely identify [56] References Cited particular leukocyte types.

UNITED STATES PATENTS 3,315,229 4/1967 Smithline 356/36 18 Clams 3 Drawmg figures IMAGE CONTROL 24 SCANN IN G TUBE 40 38 26 COMPUTER MEMORY CONTROL 22 46 IV -7 L7 35/ M r I; :7

US. Patent Oct. 28, 1975 Sheet 1 of2 3,916,205

IMAGE CONTROL FIG. I SCANNING '1 TUBE 4o COMPUTER MEMORY 38 [-30 46 CONTROL 22 F SUSPENSION 74 78 U.S. Patent Oct. 28, 1975 Sheet 2 of2 3,916,205

RATIO OF THE GREEN COMPONENT OF CELL S S m E F L m w m If E W O N H E% N O R O D.- CE E N T M M NL C l U Y 6 C 8 E L S 0 CU E N 5N w L m H H T WT R F CAL U w mm a m 4 RF U I M @v o a o 3. E 0 5 QM R O O U 0 A m %0 AA 3 D M G m AA I2 I F. R O U L F (ll IIL 0 0 5. O 5 0 a I 0 1 particular leukocyte type; I a

BACKGROUND OF THE INVENTION 1. Field of the Invention: v

The present invention relates to cytology and, more particularly is directed towards a composition for staining specific components of cells particularly blood cells,and a method and apparatus for differentially counting and classifying leukocyte types.

2. Description of the Prior Art:

Presently, there are several methods and systems for counting and classifying leukocytes. In the usual method, a cytotechnician microscopically views ablood smear prepared on an ordinary microscope slide that has been stained with one of the Romanowsky stains, such as'the Wright or Giemsa stains. The cytotechnician sequentially examines I leukocytes and classifies each accordingly to its type. Not only is this method time consuming, but it suffers also from the disadvantage of limited reliability relative to counting and classifying of the less abundant cells such as monocytes, eosinophils and basophils. In a recently developed automated system, the leukocytes are made to flow through three or four different channels, each channel provided with means for staining the leukocytes flowing therein with a particular dye. Due to the fact that several channels and difl'erent chemical treatments are required for each cell type, such a system sufiers from thedisadvantages that it is complex in design and costly to manufacture.

In another automatic system, a selected region of a blood smear, stained in the usual manner with a Romanowsky stain, is scanned mechanically under a microscope provided with an electronic image tube. When a leukocyte is in the field of view of the image tube, the slide is stopped. An image analyzing computer connected to the image tube classifies the leukocyte according to its cell profile and cytoplasm color. Since this system requires'the services of a technician to select a region of the smear suitable for automatic scanning, such systems have suffered from the disadvantages of being time consumming and costly.

SUMMARY OF THE INVENTION It is an object of the present invention to provide a composition of matter, a method and an apparatus involving the automatic absolute counting of leukocytes,

erythrocytes reticulocytes, and other cells, and the differential counting and classifying of leukocyte types which do not sufl'er from the heretofore mentioned disadvantages. The present invention provides a dye com position for distinguishing eosinophils, monocytes, lymstained leukocytes are irradiated with optical radiation having the characteristic absorption wavelengths of each dye, the relative intensities of the light emitted and/or by each leukocyte defines 'a The term compatible d'yesis used here to mean that no dye in the mixture prevents any other dye in the mixture from selectively staining characteristic structures of the cell types of interest when irradiated in the characteristic absorption region of said other dye, such selective staining being essentially not different from the action of said other dye when used by itself.

The term fluorochrome stain is used to designate a stain which under suitable illumination produces better differentiated optical parameters about the cell type of interest by its fluorescence than by its absorption properties, for instance, when the light transmitted by the dye in its main absorption band is not much different in intensity from that transmitted by its surroundings, but the fluorescence intensity of the same dye is several times or more greater than that of its surroundings. The term differential staining is used to mean that the dye being used will stain a given component of the cell type of interest for instance the protein of said cell type to a much greater extent that other components, for instance nucleic acids, of the same cell type. One fluorescent dye composition, particularly adapted for slide scanning and flow tube methods for diflerentially counting and classifying leukocytes, comprises sulfonated triazinyl derivatives of a diarnino stilbene, having each amino group in each of the phenyl rings of the stilbene molecule, preferably a sulfonated 4,4 diamino stilbene, either of the dyes 8-p-toluidino-lnaphthalene sulfonic acid or brilliant sulfaflavine, and ethidium bromide. In the slide scanning method, a drop of blood is spread on a microscope slide and, after fixation, is stained by immersion in the dye composition. In the flow tube method, a small amount of a blood sample, suitably diluted, is stained in a liquid solution of the dye composition and the cells are forced to flow in single file through a capillary tube. In both methods, the stained leukocytes are irradiated under ultraviolet and violet light and their fluorescence is detected through a blue-green filter. The leukocytes are presented with a fluorescence in decreasing order to intensity from eosiniphils to immature neutrophils to mature neutro phills to monocytes to lymphocytes. The stained leukocytes are then irradiated under a greenlight and the nuclei of the different leukocyte types are detected through a red filter. Leukocytes exhibit a red fluorescence of comparable intensity, reticulocytes fluoresce with an appreciably smaller intensity and normal erythrocytes have a negligible fluorescence. The leukocytes are counted and classified, for example, by measuring the ratio of the blue-green component of cell fluorescence to the red nuclear fluorescence. In the flow tube method, excitation with ultraviolet light produces a strong orange-red component, most pronounced in neutrophils and eosinophils, the intensity of which, with respect to the blue-green component, provides an- 7 other parameter for distinguishing particular leukocyte types. In addition, the transmitted violet light is measured to distinguish erythrocytes from leukocytes. In contrast to the negligible absorption of violet light by leukocytes, erythrocytes and reticulocytes exhibit an appreciable absorption of violet light.

The invention accordingly comprises the composition, method steps and apparatus possessing the construction, combination of elements, and arrangement of parts that are exemplified in the following detailed disclosure, the scope of which will be indicated in the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS For a fuller understanding of the nature and objects of the present invention, reference should be had to the following detailed description taken in connection with the accompanying drawings wherein:

FIG. 1 is a block and schematic diagram of a slide scanning system for differential counting of leukocytes;

FIG. 2 is a block and schematic diagram of a flow tube system for differential counting of leukocytes; and

FIG. 3 is a graphic representation illustrating the fluorescent characteristics of selected leukocyte types on a methanol fixed smear stained with one of the mixtures herein described.

DETAILED DESCRIPTION OF THE INVENTION Generally, the process embodying the present invention involves the staining of a blood medium with a dye composition comprising a plurality of dyes, at least one of the dyes imparting a characteristic fluorescence to all leukocytes. The dye composition is such that, when the stained blood medium is illuminated with light having the characteristic absorption wavelengths of each dye, the relative intensities of the light emitted and/or transmitted by each cell type at the optical wavelength region of emission and absorption characteristic of each dye uniquely depends, in at least one characteristic wavelength region, on the type of leukocyte. The light emitted by the irradiated leukocytes in each of their characteristic wavelength regions is measured and the leukocyte types are classified according tothe relative intensities of the emitted light in the characteristic wavelength region of each dye.

In particular, the process embodying the present invention involves the staining of leukocytes randomly distributed in a medium with a dye composition comprising a mixture of (l) a sulfonated triazinyl stilbene derivative, (2) a naphthalene sulfonic acid; and (3) a cationic dye. The stained leukocytes are irradiated with ultraviolet and violet light and detected through a green or blue-green filter. The green component of leukocyte fluorescence is presented with a characteristic fluorescent intensity against a dark background. The stained leukocytes are also irradiated with green light and the red fluorescence of only the nuclei of the cells are detected through a red or orange-red filter. As shown in FIG. 3, the detected leukocytes are characterized by a fluorescence in decreasing order of intensity from eosinophils to neutrophils to monocytes to lymphocytes, each leukocyte type uniquely defined by the ratio of the blue-green component of cell fluorescence to the red nuclear fluorescence with respect to the red fluorescence from the nucleus.

The dyes for protein, such as the cytoplasm of white blood cells and granules of granulocytes, preferably are sulfonated triazinyl derivatives of a diamino stilbene, for example 4,4-bis 4-(3 sulfoanilino)-6-[bis (2- hydroxy-ethyl)-amino]-l ,3,5triazin-2-yl amino stilbene 2,2disulfonic acid tetrasodium salt and their alkyl, alkoxy or halogen substituted derivatives. For these dyes, the optimum excitation wavelength band is in the range of 320 to 390 nanometers and the optimum fluorescent wavelength band is 440 to 550 nanometers. These dyes impart a strong blue fluorescence to the protein of leukocytes and the neutrophil granules and a weaker blue fluorescence to eosinophils, monocytes and lymphocytes.

The dyes for eosinophil granules preferably are the anilino or toluidino naphthalene sulfonic acids and their alkyl, alkoxy or halogen substituted derivatives;

4,4 diamino stilbene 2,2 disulfonic acid, N, N, N, N' tetraacetric acid and its alkyl, alkoxy or halogen substituted derivatives; sulfonated fluorescent derivatives of 1,8 naphthalimide, such as'brilliant s'ulfaflavine and its alkyl, alkoxy or halogen substituted derivatives; 8-ptoluidino-l naphthalene sulfonic acid andits alkyl, alkoxy or halogen substituted derivatives; and 8-hydroxyl, 3, 6 pyrene trisulfonic acid and its alkyl, alkoxy and halogen derivatives. The anilino and toluidino naphthalene sulfonic acids have an optimum excitation wavelength band in the range of 320 to 410 nanometers and an optimum fluorescent wavelength band in the range of 440 to 550 nanometers. Brilliant sulfoflavine has an optimum excitation wavelength band in the range of 360 to 450 nanometers and an optimum fluorescent wavelength band in the range of 480 to 550 nanome-- ters. These dyes strongly stain the'eosinophil granules with a green-blue or green fluorescence.

The dyes which impart a strong red fluorescence to the nucleic acids of all leukocytes, mainly in the nuclei, preferably are cationic dyes, for example the phenanthridinium dyes such as ethidium bromide and its alkyl, alkoxy or halogen derivatives; and in dry smears, acridine orange; and rhoduline orange. Ethidium bromide has an optimum excitation wavelength range of 480 to 550 nanometers and an optimum fluorescent wavelength range of 580 to 650 nanometers.

Referring now to FIG. 1, there is shown a slide scanning system 10 for differentially counting leukocytes. By way of example, an alcohol-fixed blood smear 11 on a slide 12 is immersed in a preferred dye solution comprising 1 X 10 molar ethidium bromide; l X 10' to l X 10 molar brilliant sulfaflavine; andl X 10 molar 4,4 bis 4-(3-sulfoanilino)-6[bis (2-hydroxyethyl)amino]-l,3,5 triazin-Z-yl amino stilbene 2,2 disulfonic acid tetrasodium salt in a solvent including a buffer of 0.01 to 0.1 molar 2-amino-2(hydroxymethyl)-l,3 propanediol-HCI, commonly known as tris- HCl, at a pH concentration range of 8.0 to 10.0, for best results, the pH concentration range is 8.5 to 9.5. In alternative embodiments, the buffer is other than tris-I-ICl, for example sodium borate or sodium bicarbonate. After a staining time of approximately 10 minutes, slide 12 is rinsed in an aqueous solution, for example distilled water, for approximately 1 minute and then dried.

Thereafter, slide 12 is irradiated with illumination from a source 14, for example a mercury arc lamp, which is focused thereon via a collimating lens 16, an excitation filter wheel 18, a beam splitter 20 and a condensing lens 22. Filter wheel 18 includes a filter 32 which transmits ultraviolet and violet light, for example 320 to 440 nanometers and a filter 34 which transmits green light, for example the 546 nanometer mercury band. In the illustrated embodiment, by way of example, beam splitter 20 is a dichroic mirror. In alternative embodiments, beam splitter 20 is other than a dichroic mirror, for example a reflector such as a straight surface rnirror. The field of view containing the fluorochromed leukocytes is imaged on a photo-electronic device 24, for example the photosensitive surface of an image scanning tube, via a collimating lens 26 and an emission filter wheel 28. Filter wheel 28 includes a blue-green filter 36 and a red filter 38. As hereinafter described, filter wheels 18 and 28 are indexed by means of a controller 30.

Initially, controller 30 indexes filter wheels 18 and 28 insuch a manner that filters 32 and 36 are positioned -in the transmitted light path. That is, slide 12 is irradi- ,ated with ultraviolet and violet light and blue-green fluorescent images are presented at the photosensitive .eosiniphils and neutrophils. The photosensitive surface of image tube 24 is scanned in a specified pattern determined by a control 40 which is programmed by a computer 42. The intensity of the irradiated leukocytes are measured by computer 42 and data signals for each measurement are stored in a memory 44 at X,Y address location corresponding to the X,Y positions on the photosensitive surface of image scanning tube 24.

Controller 30 then indexes filter wheels 18 and 28 in such a manner that filters 34 and 38 are positioned in the transmitted light path. That is, slide 12 is irradiated with green light and red fluorescent images are presented at the photosensitive surface of image scanning tube 24 through red filter 38. In this case, only the red fluorescence of the nuclei of the leukocytes appear against a dark background at the photosensitive surface of image tube 24; In the manner hereinbefore described, the fluorescent intensities of the nuclei at each of the X,Y positions on the photosensitive surface of image scanning tube 24 are measured by computer 42.

The measurement data stored'in memory 44, i.e., the

green component of cell fluorescence, is addressed into computer42 for determining leukocyte types by generating differential counting data signals representing the ratio of the green component of cell fluorescence to red nuclear fluorescence with respect to the red fluorescence from the nucleus as shown in FIG. 3. Cell profile data signals, i.e. ameasurement of the time period during which a signal from a cell is received, distinguish lymphocytes from monocytes. The differential counting data signals generated by computer 42 are applied to a display 46, for example a digital display of the relative abundance of the different cell types, for visual presentation. It is to be understood that, in alternative embodiments display 46 is other than a digital display, for example a cathode-ray tube, a chart recorder, or a magnetic tape recorder. 7

It will be readily appreciated that systems other than that shown in FIG. 1 can be used for differentially counting and classifying leukocytes types on a stained blood smear. For example, a manual system wherein the stained slide is irradiated in the manner hereinbefore described and observed through a microscope. In alternative embodiments, the irradiated leukocytes are detected by means other than an image scanning tube, for example one or a plurality of photo-detectors and a flying spot scanner.

Referring now to FIG. 2, there is shown a flow tube system 50 for differential counting of leukocyte types. By way of example, a blood sample is diluted in a saline solution and the cells are fixed with formaldehyde. It is to be noted that the cells can be fixed with formaldehyde prior to or after dilution. The white cells are stained in a solution comprising 1 X molar 4,4 bis I 4-(3 sulfoanilino)-6-[bis (2-hydroxy ethyl) amino]- 1,3,5 triazin -2- yl}amino stilbene; 1 X 10 to l X 10 molar brilliant sulfaflavine; and l X 10 molar ethidium bromide. The resulting suspension is diluted and buffered with either 0.1 molar tris-HCl or 0.05 molar borax at a pH in the range of 9.0 to 9.2. As an example, the blood sample has a dilution range. of fifty to one hundred fold in the suspension, the final concentration of which is approximately 1 X 10 molar or less in each of the fluorescent dyes. The cells. are made to flow single file through a narrow tube 52, for example a capillary tube, wherein each of the leukocytes is irradiated with illumination from a source 54, for example a mercury arc lamp. The illumination generated by mercury arc lamp 54 is directed through a collimating lens 56 to a reflecting filter 58, for example a dichroic filter. The wavelength band of light passing through dichroic filter 58 is focused on capillary tube 52 at 62 via a condensing lens 64. In the illustrated embodiment, by way of example, dichroic filter 58 transmits green light in the 546 nanometer mercury band. The wavelength band of light reflected by dichroic filter 58 is directed to a reflecting filter 66, for example a dichroic filter, and focused on capillary tube 52 at via a condensing lens 72. In the illustrated embodiment, by way of example, dichroic filter 66 reflects ultraviolet and violet light in the 320 to 410 nanometer mercury band. It is preferred that the distance between the locations denoted by reference characters 62 and 70 is typically in the range of 50 to 200 microns. A photo-electric device 74, for example a photo-multiplier senses the blue-green emission light from capillarytube 52 via a collecting lens 76, a dichroic mirror 77 and a blue-green emission filter 78. A photo-electric device 80, for example a photo-multiplier, senses the orange-red emission light from capillary tube 52 via collecting lens 76, dichroic mirror 77, a dichroic mirror 82 and an orange-red emission filter 84. A photo-electric device 81, for example a photodiode, senses the ultraviolet and violet light at 70 via an emission filter 82 having a pass band in the approximaterange of 410 to 430 nanometers.

Photo-multiplier 74 detects the blue-green component of cell fluorescence, and photo-multiplier 80 detects the red fluorescence, and photo-multiplier 81 detects the violet light. Erythrocytes and reticulocytes are characterized by appreciable absorption of violet light and leukocytes are characterized by negligible absorption of violet light. Accordingly, photo-multiplier 81 detects the violet light absorption for distinguishing red cells from white cells.

Data signals generated by photo-multipliers 74 and 80 are applied to a precessor 86, for example a small dedicated computer, wherein the detected leukocyte fluorescences are differentially counted and classified as the ratio of the green component of cell fluorescence to the red nuclear fluorescence with respect to the red fluorescence from the nucleus for each leukocyte type. Differential counting data signals generated by computer 86 for each leukocyte passing through capillary of photodetectors, for example photodiodes. It is to be understood that, in alternative embodiments, display 88 is other than a digital display for example, a cathode-ray tube, a chart recorder or a magnetic tape recorder. Furthermore, it is to be understood that, in alternative embodiments, the light emitted from source 54 is directed to tube 52 by means other than a pair of beam splitters, for example, two sources each emitting light which is directed towards tube 52 or a source characterized by a scanning light beam.

The following non limiting examples further illustrate the staining of slides and suspensions hereinbefore described.

EXAMPLE I SLIDE A drop of whole blood is placed on a microscope slide and spread into a thin film with the aid of another microscope slide. After the film has dried, the blood smear is fixed by immersing the slide in methyl alcohol for five minutes. Thereafter, the slide is immersed in a liquid mixture comprising:

1. 100 parts per million of 4,4'-bis 4-(3 sulfoanilino)-6-[bis (2-hydroxy-ethyl)-amino]-l,3,5 triazin-Z-yl amino stilbene 2,2-disulfonic acid tetrasodium salt;

2. 1,000 parts per million of brilliant sulfaflavine; and

3. 30 parts per million of ethidium bromide;

4. 12,000 parts per million of 2-amino-2-(hydroxymethyl)-1,3 propanediol and enough concentrated hydrochloric acid solution to bring the pH of the solution to 9.0.

After ten minutes in the liquid mixture, the slide is rinsed in distilled water for one minute and then dried.

EXAMPLE I] SUSPENSION One part of whole blood is mixed with four parts of a solution at a pH of 9.0 comprising:

1. 1,200 parts per million of 2-amino-2-(hydroxymethyl)-l,3 propanediol-HCI buffer pH 9.0;

2. 85,000 parts per million of sodium chloride;

3. 1,000 parts per million of brilliant sulfaflavine;

4. 100 parts per million of 4,4-bis 4-(3- sulfoanilino)-6-[bis (2-hydroxyethyl)-amino]- 1,3,5triazin-2-yl amino stilbene 2,2-disulfonic acid tetrasodium salt; and

5. 30 parts per million of ethidium bromide.

After three minutes, the suspension is mixed with one part of a 20% solution of formaldehyde. Five minutes later, the mixture is diluted 15 to 20 times with a solution comprising:

6. 12,000 parts per million of 2-arnino-2-(hydroxymethyl)-l,3 propanediol-HCI buffer (pH 9.0);

7. 85,000 parts per million of sodium chloride;

8. 100 parts per million of 4,4'-bis 4-(3- sulfoanilino)-6-[bis (2-hydroxy-ethyl)-amino]- 1,3,5 triazin-2-yl amino stilbene 2,2'-disulfonic acid tetrasodium salt; and

9. 15 parts per million of ethidium bromide.

In this example, the stained suspended cells exhibit the following optical properties under ultraviolet illumination of approximately 365 nanometers:

a. The cytoplasm and granules of the neutrophils exhibit a visible fluorescence with spectral peaks in the blue and orange-red regions. The blue component results from the direct excitation of the triazinyl dye and the red component, most pronounced in the granules, is due to energy transfer from the triazinyl dye to ethidium bromide, the latter being present in the granules, and elsewhere in the cytoplasm, at concentrations which are too small to be efficienctly excited by direct absorption of the ultraviolet light. b. The cytoplasm and granules of the eosinophils exhibit a stronger visible fluorescence than the neutrophils with spectral peaks in the blue-green and orange-red regions. The blue-green component results mainly from the excitation of the brilliant sulfaflavine dye and the triazinyl dye, and the redorange component is due to energy transfer from the triazinyl dye and the brilliant sulfaflavine dye to ethidium bromide; the latter being present in the granules, and elsewhere in the cytoplasm at concentrations too small to be efficiently excited by direct absorption of the ultraviolet light.

The cytoplasm and nucleus of lymphocytes and monocytes exhibit a weaker fluorescence than that of either the neutrophils or eosinophils. This fluorescence is characterized by a spectral distribution having a peak in the blue region and a smaller peak or shoulder in the red region, the fluorescence of the monocytes being smaller than the fluorescence of the lymphocytes.

Under violet illumination of approximately 400 to 440 nanometers, the eosinophils exhibit a strong green fluorescence which is several times stronger than that of the other leukocytes.

Under green illumination, the nuclei, and to a lesser extent the cytoplasm, of all the leukocytes exhibit a red fluorescence; eosinophils having the brightest fluorescent intensity, neutrophils and monocytes having comparable fluorescent intensities of a lesser brilliance, and

lymphocytes generally having the lowest fluorescent intensity.

It is to be noted that red cells exhibit a negligible fluorescence under any of the above conditions.

EXAMPLE III SUSPENSION One part of whole blood is mixed with one part of a 10% solution of formaldehyde for five minutes. The suspension is then mixed with four parts of a solution comprising:

1. 1,200 parts per million of 2-amino-2-(hydroxymethyl)-l,3 propanediol-H01 buffer (pH 9.0);

2. 85,000 parts per million of sodium chloride;

3. 300 parts per million of 8-p toluidino-lnaphthalene sulfonic acid;

parts per million of 4,4'-bis 4-(3- sulfoanilino )-6-[bis (2-hydroxy-ethyl)-amino]- 1,3,5 triazin-Z-yl amino stilbene 2,2'-sulfonic acid tetrasodium salt; and

5. 20 parts per million of ethidium bromide.

After five minutes, the mixture is diluted 15 to 20 times with a solution comprising:

6. 12,000 parts per million of 2-amino-2-(hydroxymethyl)-l,3 propanediol-HCl buffer (pH 9.0);

7. 85,000 parts per million of sodium chloride;

8. 60 parts per million of 8 toluidino l naphthalene sulfonic acid;

100 parts per million of 4,4'-bis (3sulfoanilino)-6-[bis(2-hydroxy-ethyl)-amino]- 9 1,3,5 triazin-2 -yl amino stilbene2,2-disulfonic acid tetrasodium salt; and 10. 15 parts per million of ethidium bromide. In this example, the stained suspended cells exhibit the following optical properties under ultraviolet illumination of approximately 365 nanometers:

a. The cytoplasm and granules of the neutrophils ex-' hibit a visible fluorescence with spectral peaks in the blue and orange-red regions. The blue component results from the direct excitation of the triazinyl dye and the red component, most pronounced in the granules, is due to energy transfer from the triazinyl dye to ethidium bromide, the latter being present in the granules, and elsewhere in the cytoplasm, at concentrations which are too small to be efficiently excited by direct absorption of the ultraviolet light. b. The cytoplasm and granules of the eosinophils exhibit a stronger visible fluorescence than the neutrophils with spectral peaks in the blue-green and orange-red regions. The blue-green component results mainly from the excitation of 8-p-toluidino-lnaphthalene sulfonic acid and the triazinyl dye, and the red-orange component is due to energy transfer from the triazinyl dye and the 8-ptoluidino-lnaphthalene sulfonic acid to ethidium bromide; the latter being present in the granules, and elsewhere in the cytoplasm at concetrations too small to be efficiently excited by direct absorption of the ultraviolet light.

The cytoplasm and nuclei of lymphocytes and monocytes exhibit a weaker fluorescence than that of either the neutrophils or eosinophils. This fluorescence is characterized by a spectral distribution having a peak in the blue region and a smaller peak or shoulder in the red region, the fluorescence of the monocytes being smaller than the fluorescence of the lymphocytes.

Under violet illumination of approximately 400 to 440 nanometers, the eosinophils exhibit a strong green fluorescence which is several times stronger than that of the other leukocytes.

Under green illumination, the nuclei, and to a lesser extent the cytoplasm, of all the leukocytes exhibit a red fluorescence; eosinophils having the brightest fluorescent intensity, neutrophils and monocytes having comparable fluorescent intensities of a lesser brilliance, and lymphocytes generally having the lowest fluorescent intensity.

It is to be noted that red cells exhibit a negligible fluorescenceunder any of the above conditions.

In summary, the present invention provides a single dye composition comprising three fluorescent dyes for uniquely distinguishing leukocytes, erythrocytes and reticulocytes randomly distributed in a medium and a method and apparatus for absolute counting of leukocytes and reticulocytes and differential counting of leukocytes which have been stained with such a dye composition. When the stained leukocytes are irradiated by light having the characteristic absorption wavelengths of each dye, the relative intensities of leukocyte fluorescence at the optical wavelength region of emission of each dye depends, in at least one characteristic wavelength region, on the type of leukocyte. The light emitted by the irradiated leukocytes in each of their characteristic wavel'ength regions is measured and the leukocyte types are classified according to the relative intensities of the emitted light in the characteristic wavelength region of each dye. Transmitted violet light is measured to distinguish erythrocytes, reticulocytes and leukocytes, erythrocytes and reticulocytes exhibiting appreciable absorption of violet light and leukocytes exhibiting negligible absorption of violet light.

Since certain changes may be made in the foregoing disclosure without departing from the scope of herein involved, it is intended that all matter contained in the above description and depicted in the accompanying drawings be construed in an illustrative and not in a limiting sense. i

What is claimed is: 1. Method of identifying a blood cell, and comprising the steps of:

staining said blood cell with a plurality of dyes, at

least one of which is a fluoresecent dye; illuminating the dyed cell with a sequence of expo sures to radiation, each of said exposures being at a limited wavelength band corresponding to the wavelength absorption band of a respective one of said dyes;

observing during each such exposure the characterisv I tic wavelength band of emission or transmission by a selected one of said dyes in said dyed cell in response to each of said exposures; and

comparing'with one another the relative intensities of the emission or transmission observed for each sequential exposure, whereby said cell can be characterized by such comparison.

2. Mehtod as defined in claim 1 wherein another'dye of saidplurality of dyes is a sensitizer capable of transferring energy absorbed in its characteristic absorption wavelength band to molecules of, said fluorescent dye in proximity to said sensitizer; and,

further including the step of observing the fluorescent emission from said dyed cell in the characteristic wavelength emission band of said fluorescent dye in response to an exposure of said dyed cell to radiation in the characteristic wavelength absorption band of said sensitizer.

3. A method as defined in claim 1 wherein said plurality is at least three, and one of said dyes differentially dyes nucleic acids of said cell, one of said dyes stains the basic portions of said cell and the third of said dyes dyes the acidic portions of said cell.

4. A method as defined in claim 1 wherein at least two of said dyes are fluorescent dyes which dye different types of blood cells so that each said type fluoresces under each said illumination with a different intensity.

5. A method as defined in claim 4 wherein said cell types are leukocytes, erythrocytes and reticulocytes.

6. A method as defined in claim 4 wherein said cell types are different types of leukocytes.

7. The method for identifying leukocyte types as claimed in claim 6 wherein said plurality of dyes includes brilliant sulfalfavine for imparting a blue-green fluorescence to eosinophil granules.

10. The method for identifying leukocyte types as claimed in claim 6 wherein said plurality of dyes includes ethidium bromide for imparting a red fluorescence to the nuclei of all. leukocytes.

1 1. A system for identifying leukocytes, erythrocytes and reticulocytes randomly distributed in a medium, said system comprising:

a. means for staining said leukocytes, erythrocytes and reticulocytes with a dye composition including sulfonated triazinyl derivatives of 4,4 diamino stilbene, sulfonated fluorescent derivatives of 1,8 napthalimide, and a phenanthridinium dye;

b. source means for sequentially illuminating the stained leukocytes, erythrocytes and reticulocytes with optical radiation in the characteristic absorption wavelength band of each said dye and c. detector means optically coupled to said irradiated leukocytes, erythrocytes and reticulocytes for identifying each said type cell according to its fluorescent intensity.

12. The system as claimed in claim 11 wherein said sulfonated fluorescent derivatives of 1,8 naphthalimide is brilliant sulfaflavine.

13. The system as claimed in claim 11 wherein said phenanthridinium dye is ethidium bromide.

14. The system as claimed in claim 11 wherein said detector means includes photodetector means for measuring light scattered by said leukocytes for differentiating said leukocytes according to their size.

15. A system for differentially classifying and counting leukocyte types, particularly eosinophils, neutrophils, monocytes and lymphocytes stained with at least a pair of compatible fluorescent dyes and fixed on slide means, said system comprising:

means for illuminating said slide means with a sequence of at least two exposures, each of said expo sures being at a different wavelength band corresponding to the wavelength absorption band of a corresponding one of said dyes;

detector means optically coupled to said slide means for detecting, during each of said exposures, the characteristic wavelength band emitted by one of said dyes in said leukocytes in response to a corresponding one of said exposuresbut not the characteristic wavelength band emitted by any of the other dyes responsively to said one of said exposures; and

means responsive to said detector means for classifying and counting said leukocyte types according to a comparison of the fluorescent intensities emitted by each said leukocyte type during a sequence of exposures.

16. A system for differentially classifying and counting leukocyte types randomly distributed in a blood sample, said system comprising:

means for staining the leukocyte types in said sample with a dye composition comprising a plurality of fluorescent dyes, at least one of said fluorescent dyes imparting a first characteristic fluorescence to the nuclei of all said leukocytes, said other fluorescent dyes imparting a second characteristic fluorescence to the cytoplasm of eosinophils, neutrophils, monocytes and lymphocytes;

means for flowing the stained leukocytes in said sample in single file; source means for illuminating said stained leukocytes passing in single file through said flow means with .a sequence of at least two exposures, each of said exposures being at a different wavelength band corresponding to the wavelength absorption band of a corresponding one of said dyes; detector means optically coupled to said flow means for detecting, during each of said exposures, only the characteristic wavelength band emitted by one of said dyes in said leukocytes in response to a corresponding one of said exposures but not any wavelength band emitted by any other of said dyes in response to said one of said exposures; and means responsive to said detector means for classifying and counting said leukocyte types according to a comparison of the fluorescent intensities emitted by each said leukocyte type during a sequence of exposures. 17. System for identifying a blood cell stained with a plurality of compatible dyes, at least one of which is a fluorescent dye, said system comprising, in combination:

means for illuminating said cell with a sequence of exposures to radiation, each exposure of said sequence being at a different wavelength band corresponding to the wavelength absorption band of a corresponding one of said dyes; means for observing during each of said exposures only the characteristic wavelength band emitted or transmitted by one of said dyes in said cell in response to a corresponding one of said exposures;

means for generating a signal in response to each emission or transmission from said cell resulting from each of said exposures; and

means for comparing said signals to obtain a characteristic value for said cell.

18. System as defined in claim 17 wherein said means for illuminating includes means for providing at least two of said exposures at different wavelength bands corresponding to the absorption bands of respective ones of said dyes; and i said means for observing includes means for observing fluorescent emission in the wavelength band of emission from said fluorescent dye dueonly to exposure of said cell to radiation in the wavelength absorption band of said fluorescent dye, and means for observing fluorescent emission in the wavelength band of emission from said fluorescent dye due only to exposure of said cell to radiation in the wavelength absorption band of another of said dyes. 

1. Method of identifying a blood cell, and comprising the steps of: staining said blood cell with a plurality of dyes, at least one of which is a fluoresecent dye; illuminating the dyed cell with a sequence of exposures to radiation, each of said exposures being at a limited wavelength band corresponding to the wavelength absorption band of a respective one of said dyes; observing during each such exposure the characteristic wavelength band of emission or transmission by a selected one of said dyes in said dyed cell in response to each of said exposures; and comparing with one another the relative intensities of the emission or transmission observed for each sequential exposure, whereby said cell can be characterized by such comparison.
 2. Method as defined in claim 1 wherein another dye of said plurality of dyes is a sensitizer capable of transferring energy absorbed in its characteristic absorption wavelength band to molecules of said fluorescent dye in proximity to said sensitizer; and further including the step of observing the fluorescent emission from said dyed cell in the characteristic wavelength emission band of said fluorescent dye in response to an exposure of said dyed cell to radiation in the characteristic wavelength absorption band of said sensitizer.
 3. A method as defined in claim 1 wherein said plurality is at least three, and one of said dyes differentially dyes nucleic acids of said cell, one of said dyes stains the basic portions of said cell and the third of said dyes dyes the acidic portions of said cell.
 4. A method as defined in claim 1 wherein at least two of said dyes are fluorescent dyes which dye different types of blood cells so that each said type fluoresces under each said illumination with a different intensity.
 5. A method as defined in claim 4 wherein said cell types are leukocytes, erythrocytes and reticulocytes.
 6. A method as defined in claim 4 wherein said cell types are different types of leukocytes.
 7. The method for identifying leukocyte types as claimed in claim 6 wherein said plurality of dyes includes sulfonated triazinyl derivatives of 4,4'' diamino stilbene for imparting a blue fluorescence to the protein of leukocytes and to neutrophil and eosinophil granules.
 8. The method for identifying leukocytes types as claimed in claim 6 wherein said plurality of dyes includes 8-p-toluidino 1-naphthalene sulfonic acid for imparting a blue-green fluorescence to eosinophil granules.
 9. The method for identifying leukocytes distributed as claimed in claim 6 wherein said plurality of dyes includes brilliant sulfalfavine for imparting a blue-green fluorescence to eoSinophil granules.
 10. The method for identifying leukocyte types as claimed in claim 6 wherein said plurality of dyes includes ethidium bromide for imparting a red fluorescence to the nuclei of all leukocytes.
 11. A system for identifying leukocytes, erythrocytes and reticulocytes randomly distributed in a medium, said system comprising: a. means for staining said leukocytes, erythrocytes and reticulocytes with a dye composition including sulfonated triazinyl derivatives of 4,4'' diamino stilbene, sulfonated fluorescent derivatives of 1,8 napthalimide, and a phenanthridinium dye; b. source means for sequentially illuminating the stained leukocytes, erythrocytes and reticulocytes with optical radiation in the characteristic absorption wavelength band of each said dye and c. detector means optically coupled to said irradiated leukocytes, erythrocytes and reticulocytes for identifying each said type cell according to its fluorescent intensity.
 12. The system as claimed in claim 11 wherein said sulfonated fluorescent derivatives of 1,8 naphthalimide is brilliant sulfaflavine.
 13. The system as claimed in claim 11 wherein said phenanthridinium dye is ethidium bromide.
 14. The system as claimed in claim 11 wherein said detector means includes photodetector means for measuring light scattered by said leukocytes for differentiating said leukocytes according to their size.
 15. A system for differentially classifying and counting leukocyte types, particularly eosinophils, neutrophils, monocytes and lymphocytes stained with at least a pair of compatible fluorescent dyes and fixed on slide means, said system comprising: means for illuminating said slide means with a sequence of at least two exposures, each of said exposures being at a different wavelength band corresponding to the wavelength absorption band of a corresponding one of said dyes; detector means optically coupled to said slide means for detecting, during each of said exposures, the characteristic wavelength band emitted by one of said dyes in said leukocytes in response to a corresponding one of said exposuresbut not the characteristic wavelength band emitted by any of the other dyes responsively to said one of said exposures; and means responsive to said detector means for classifying and counting said leukocyte types according to a comparison of the fluorescent intensities emitted by each said leukocyte type during a sequence of exposures.
 16. A system for differentially classifying and counting leukocyte types randomly distributed in a blood sample, said system comprising: means for staining the leukocyte types in said sample with a dye composition comprising a plurality of fluorescent dyes, at least one of said fluorescent dyes imparting a first characteristic fluorescence to the nuclei of all said leukocytes, said other fluorescent dyes imparting a second characteristic fluorescence to the cytoplasm of eosinophils, neutrophils, monocytes and lymphocytes; means for flowing the stained leukocytes in said sample in single file; source means for illuminating said stained leukocytes passing in single file through said flow means with a sequence of at least two exposures, each of said exposures being at a different wavelength band corresponding to the wavelength absorption band of a corresponding one of said dyes; detector means optically coupled to said flow means for detecting, during each of said exposures, only the characteristic wavelength band emitted by one of said dyes in said leukocytes in response to a corresponding one of said exposures but not any wavelength band emitted by any other of said dyes in response to said one of said exposures; and means responsive to said detector means for classifying and counting said leukocyte types according to a comparison of the fluorescent intensities emitted by each said leukocyte type during a sequence of exposures.
 17. System for identifying a blood cell stained with a plurality of compatible dyes, at least one of which is a fluorescent dye, said system comprising, in combination: means for illuminating said cell with a sequence of exposures to radiation, each exposure of said sequence being at a different wavelength band corresponding to the wavelength absorption band of a corresponding one of said dyes; means for observing during each of said exposures only the characteristic wavelength band emitted or transmitted by one of said dyes in said cell in response to a corresponding one of said exposures; means for generating a signal in response to each emission or transmission from said cell resulting from each of said exposures; and means for comparing said signals to obtain a characteristic value for said cell.
 18. System as defined in claim 17 wherein said means for illuminating includes means for providing at least two of said exposures at different wavelength bands corresponding to the absorption bands of respective ones of said dyes; and said means for observing includes means for observing fluorescent emission in the wavelength band of emission from said fluorescent dye due only to exposure of said cell to radiation in the wavelength absorption band of said fluorescent dye, and means for observing fluorescent emission in the wavelength band of emission from said fluorescent dye due only to exposure of said cell to radiation in the wavelength absorption band of another of said dyes. 